1. Field of the Invention
The present invention relates to reagent compositions containing an enzyme exhibiting lecithin:cholesterol acyl transferase (LCAT) activity and to elements and methods for the use of such compositions in assaying for total cholesterol in liquids.
2. Discussion Relative to the Prior Art
Cholesterol is present in biological aqueous liquids such as serum or plasma, partially in free form and partially in the form of various cholesterol esters. Known quantitative analyses of total cholesterol (i.e., the sum of the cholesterol contributions from free and esterified cholesterol) include the use of corrosive chemicals to hydrolyze the cholesterol esters to free cholesterol followed by the analysis of the total free cholesterol in solution. In one conventional technique, the desired blood serum components are extracted with an organic solvent, the cholesterol esters are saponified with alcoholic KOH, and free cholesterol is isolated and assayed. In the assay, corrosive chemicals such as ferric perchlorate and sulfuric acid are employed.
More recently, enzymes have been used to convert cholesterol and cholesterol esters into detectable products. These processes usually entail the initial conversion of cholesterol esters to free cholesterol using cholesterol esterase enzymes, also referred to as cholesterol ester hydrolase enzymes. The use of cholesterol esterase enzymes in this manner is described in U.S. Pat. Nos. 3,869,349 issued Mar. 4, 1975, to Goodhue et al, and 3,983,005 issued Sept. 28, 1976, to Goodhue et al. These enzymes exhibit specific activity toward cholesterol esters to promote hydrolysis of the esters to cholesterol in aqueous medium. The terms "esterase" or "ester hydrolase" refer to an enzyme which catalyzes an hydrolysis reaction where water molecules react with the ester portion of the cholesterol ester molecule in the presence of the enzyme.
During or after esterase-promoted hydrolysis of the cholesterol ester, free cholesterol is converted to cholestenone and hydrogen peroxide in the presence of oxygen and cholesterol oxidase, and the hydrogen peroxide is detected by coupling with a dye precursor in the presence of peroxidase. These steps are well-known in the art.
Mammalian plasma is known to contain an enzyme characterized as lecithin:cholesterol acyl transferase (LCAT). It is postulated that this enzyme facilitates the transport of cholesterol from peripheral cells to the liver. The mechanism by which this occurs, as described by Glomset, J. A., J.Lipid Res., 9:155-167, 1968, entails fatty acid acyl transfer from lecithin to cholesterol in the presence of LCAT as expressed by the following reaction: ##STR1##
The propensity of LCAT to promote acyl transfer from lecithin to cholesterol has also been reported in British Pat. No. 1,501,561 published Feb. 15, 1978, as useful in the measurement of LCAT in serum. In this method, an assay medium composed of a surfactant and lecithin substrate solution is mixed with a test sample containing free cholesterol and serum level amounts of LCAT, and the mixture is incubated. LCAT activity is thereafter quantitated by colorimetrically detecting the disappearance of cholesterol in the mixture. In particular, after the assay mixture is incubated for 2-4 hours, it is treated with cholesterol oxidase, peroxidase and dye precursor to produce color in proportion to the amount of free cholesterol remaining after incubation. This procedure is based on the disappearance of cholesterol by conversion to cholesterol esters. Esters in the original sample are not converted to appreciable amounts, if any, of free cholesterol and their total quantity thus remains unknown. The patent does not teach nor suggest that LCAT can be employed to quantitate total cholesterol.
Bacterial enzymes exhibiting LCAT activity have been isolated from microorganisms. For example, it is reported that culture supernatants of Aeromonas hydrophila are enriched with a glycerophospholipid:cholesterol acyltransferase (GCAT), (MacIntyre, S., Buckley, J. T., J. Bacteriol., 135: 402-407, 1978). The enzyme is said to deacylate human erythrocyte membrane glycerophospholipids--as LCAT does--resulting in the production of cholesterol esters. Other bacterial enzymes exhibiting LCAT-like activity include those isolated from the family Vibrionaceae and Staphylococcus aureus (MacIntyre, S., Trust, T. J., Buckley, J. T., J. Bacteriol., 139: 132-136, 1979). The literature references pertaining to serum LCAT and to GCAT from microorganisms refer to the mechanism of these enzymes as applicable only to the conversion of cholesterol to cholesterol esters. That these enzymes can be employed in a reverse manner, i.e., to convert cholesterol esters to cholesterol, facilitating the quantitative determination of total cholesterol, is not reported nor suggested in the cited literature.